A Step-by-Step Guide to Sequencing and Assembly of Complete Bacterial Genomes Using the Oxford Nanopore MinION.

The Oxford Nanopore (ONT) MinION enables sequencing of longer DNA/RNA fragments compared to other sequencers, such as Illumina, etc. This nanopore method provides distinct advantages for generating complete genome assemblies from microorganisms. Specifically, the R9.

Temporal changes in the mouse gut bacteriota influenced by host sex, diet, and exercise.

The gut microbiota plays an important role in host physiology. However, the effects of host sex, lifestyle, and temporal influences on the bacterial community within the gut remain ill-defined. To address this gap, we evaluated 56 male and female mice over a 10-wk study to assess the effects of sex, diet, and exercise on gut community dynamics.

Genome analysis and description of Tunturibacter gen. nov. expands the diversity of Terriglobia in tundra soils.

Increased temperatures in Arctic tundra ecosystems are leading to higher microbial respiration rates of soil organic matter, resulting in the release of carbon dioxide and methane. To understand the effects of this microbial activity, it is important to better characterize the diverse microbial communities in Arctic soil. Our goal is to refine our understanding of the phylogenetic diversity of Terriglobia, a common but elusive group within the Acidobacteriota phylum.

Characterization and DNA Stable-Isotope Probing of Methanotrophic Bioaerosols.

The growth and activity of bacteria have been extensively studied in nearly every environment on Earth, but there have been limited studies focusing on the air. Suspended bacteria (outside of water droplets) may stay in the atmosphere for time frames that could allow for growth on volatile compounds, including the potent greenhouse gas methane. We investigated the ability of aerosolized methanotrophic bacteria to grow on methane in the airborne state in rotating gas-phase bioreactors.

Desulfoluna spp. form a cosmopolitan group of anaerobic dehalogenating bacteria widely distributed in marine sponges.

Host-specific microbial communities thrive within sponge tissues and this association between sponge and associated microbiota may be driven by the organohalogen chemistry of the sponge animal. Several sponge species produce diverse organobromine secondary metabolites (e.g.

A ribosomal operon database and MegaBLAST settings for strain-level resolution of microbiomes.

Current methods to characterize microbial communities generally employ sequencing of the 16S rRNA gene (<500 bp) with high accuracy (∼99%) but limited phylogenetic resolution. However, long-read sequencing now allows for the profiling of near-full-length ribosomal operons (16S-ITS-23S rRNA genes) on platforms such as the Oxford Nanopore MinION. Here, we describe an rRNA operon database with >300 ,000 entries, representing >10 ,000 prokaryotic species and ∼ 150, 000 strains.

Characterization of Microplastic-Associated Biofilm Development along a Freshwater-Estuarine Gradient.

Microplastic contamination is an increasing concern worldwide. Biofilms rapidly develop on surfaces in aquatic habitats, but the processes of biofilm formation and variation in bacterial community succession on different microplastics introduced into freshwater and estuarine environments are not well understood. In this study, the biofilm bacterial communities that developed on three different types of microplastics that are prevalent in the environment, high-density polyethylene (HDPE), polyethylene terephthalate (PET), and polystyrene (PS), was investigated.

Lactobacillus rhamnosus GG modifies the metabolome of pathobionts in gnotobiotic mice.

Background: Lactobacillus rhamnosus GG (LGG) is the most widely used probiotic, but the mechanisms underlying its beneficial effects remain unresolved. Previous studies typically inoculated LGG in hosts with established gut microbiota, limiting the understanding of specific impacts of LGG on host due to numerous interactions among LGG, commensal microbes, and the host. There has been a scarcity of studies that used gnotobiotic animals to elucidate LGG-host interaction, in particular for gaining specific insights about how it modifies the metabolome.

Is Oxford Nanopore sequencing ready for analyzing complex microbiomes?

This minireview will discuss the improvements in Oxford Nanopore (Oxford; sequencing technology that make the MinION a viable platform for microbial ecology studies. Specific issues being addressed are the increase in sequence accuracy from 65 to 96.5% during the last 5 years, the ability to obtain a quantifiable/predictive signal from the MinION with respect to target molecule abundance, simple-to-use GUI-based pathways for data analysis and the modest additional equipment needs for sequencing in the field.

Arctic tundra soil bacterial communities active at subzero temperatures detected by stable isotope probing.

Arctic soils store vast amounts of carbon and are subject to intense climate change. While the effects of thaw on the composition and activities of Arctic tundra microorganisms has been examined extensively, little is known about the consequences of temperature fluctuations within the subzero range in seasonally frozen or permafrost soils. This study identified tundra soil bacteria active at subzero temperatures using stable isotope probing (SIP).

Identification of a Chlorodibenzo--dioxin Dechlorinating by Stable Isotope Probing.

Polychlorinated dibenzo--dioxins (PCDDs) are released into the environment from a variety of both anthropogenic and natural sources. While highly chlorinated dibenzo--dioxins are persistent under oxic conditions, in anoxic environments, these organohalogens can be reductively dechlorinated to less chlorinated compounds that are then more amenable to subsequent aerobic degradation. Identifying the microorganisms responsible for dechlorination is an important step in developing bioremediation approaches.

Offline Next Generation Metagenomics Sequence Analysis Using MinION Detection Software (MINDS).

Field laboratories interested in using the MinION often need the internet to perform sample analysis. Thus, the lack of internet connectivity in resource-limited or remote locations renders downstream analysis problematic, resulting in a lack of sample identification in the field. Due to this dependency, field samples are generally transported back to the lab for analysis where internet availability for downstream analysis is available.

Sulfate-Reducing Naphthalene Degraders Are Picky Eaters.

Polycyclic aromatic hydrocarbons (PAHs) are common organic contaminants found in anoxic environments. The capacity for PAH biodegradation in unimpacted environments, however, has been understudied. Here we investigate the enrichment, selection, and sustainability of a microbial community from a pristine environment on naphthalene as the only amended carbon source.

Discerning autotrophy, mixotrophy and heterotrophy in marine TACK archaea from the North Atlantic.

DNA stable isotope probing (SIP) was used to track the uptake of organic and inorganic carbon sources for TACK archaea (Thaumarchaeota/Aigarchaeota/Crenarchaeota/Korarchaeota) on a cruise of opportunity in the North Atlantic. Due to water limitations, duplicate samples from the deep photic (60-115 m), the mesopelagic zones (local oxygen minimum; 215-835 m) and the bathypelagic zone (2085-2835 m) were amended with various combinations of 12C- or 13C-acetate/urea/bicarbonate to assess cellular carbon acquisition. The SIP results indicated the majority of TACK archaeal operational taxonomic units (OTUs) incorporated 13C from acetate and/or urea into newly synthesized DNA within 48 h.

Profiling bacterial communities by MinION sequencing of ribosomal operons.

Background: An approach utilizing the long-read capability of the Oxford Nanopore MinION to rapidly sequence bacterial ribosomal operons of complex natural communities was developed. Microbial fingerprinting employs domain-specific forward primers (16S rRNA subunit), reverse primers (23S rRNA subunit), and a high-fidelity Taq polymerase with proofreading capabilities. Amplicons contained both ribosomal subunits for broad-based phylogenetic assignment (~ 3900 bp of sequence), plus the intergenic spacer (ITS) region (~ 300 bp) for potential strain-specific identification.

Forensic Analysis of Polychlorinated Dibenzo-p-Dioxin and Furan Fingerprints to Elucidate Dechlorination Pathways.

Polychlorinated dibenzo-p-dioxins and -furans (PCDD/Fs) are persistent organic pollutants whose main removal process in the environment is due to biodegradation, and particularly anaerobic reductive dechlorination. Since PCDD/F congeners that are substituted in the lateral 2, 3, 7, and 8 positions are the most toxic, removal of these chlorines is advantageous, but previous studies have only demonstrated their removal under laboratory conditions. We evaluated a concentration data set of PCDD/F congeners with four or more chlorines along with all 209 polychlorinated biphenyl (PCB) congeners in surface water, treated and untreated wastewater, landfill leachate, and biosolids (NY CARP data set) to determine whether peri and peri/lateral dechlorination of PCDD/Fs occurs in these environments.

Novel reductive dehalogenases from the marine sponge associated bacterium Desulfoluna spongiiphila.

Desulfoluna spongiiphila strain AA1 is an organohalide respiring bacterium, isolated from the marine sponge Aplysina aerophoba, that can use brominated and iodinated phenols, in addition to sulfate and thiosulfate as terminal electron acceptors. The genome of Desulfoluna spongiiphila strain AA1 is approximately 6.5 Mb.

The subzero microbiome: microbial activity in frozen and thawing soils.

Most of the Earth's biosphere is characterized by low temperatures (<5°C) and cold-adapted microorganisms are widespread. These psychrophiles have evolved a complex range of adaptations of all cellular constituents to counteract the potentially deleterious effects of low kinetic energy environments and the freezing of water. Microbial life continues into the subzero temperature range, and this activity contributes to carbon and nitrogen flux in and out of ecosystems, ultimately affecting global processes.

The Effect of Diet and Exercise on Intestinal Integrity and Microbial Diversity in Mice.

Background: The gut microbiota is now known to play an important role contributing to inflammatory-based chronic diseases. This study examined intestinal integrity/inflammation and the gut microbial communities in sedentary and exercising mice presented with a normal or high-fat diet.

Methods: Thirty-six, 6-week old C57BL/6NTac male mice were fed a normal or high-fat diet for 12-weeks and randomly assigned to exercise or sedentary groups.

Identification of a Ruminococcaceae Species as the Methyl tert-Butyl Ether (MTBE) Degrading Bacterium in a Methanogenic Consortium.

The widespread use of methyl tert-butyl ether (MTBE) has caused major contamination of groundwater sources and is a concern due to its taste and odor problems, as well as its toxicity. MTBE can be degraded anaerobically which makes bioremediation of contaminated aquifers a potential solution. Nevertheless, the organisms and mechanisms that are responsible for anaerobic MTBE degradation are still unknown.

BioDry: An Inexpensive, Low-Power Method to Preserve Aquatic Microbial Biomass at Room Temperature.

This report describes BioDry (patent pending), a method for reliably preserving the biomolecules associated with aquatic microbial biomass samples, without the need of hazardous materials (e.g. liquid nitrogen, preservatives, etc.

Identification of Bacteria Synthesizing Ribosomal RNA in Response to Uranium Addition During Biostimulation at the Rifle, CO Integrated Field Research Site.

Understanding which organisms are capable of reducing uranium at historically contaminated sites provides crucial information needed to evaluate treatment options and outcomes. One approach is determination of the bacteria which directly respond to uranium addition. In this study, uranium amendments were made to groundwater samples from a site of ongoing biostimulation with acetate.

Identification of Anaerobic Aniline-Degrading Bacteria at a Contaminated Industrial Site.

Anaerobic aniline biodegradation was investigated under different electron-accepting conditions using contaminated canal and groundwater aquifer sediments from an industrial site. Aniline loss was observed in nitrate- and sulfate-amended microcosms and in microcosms established to promote methanogenic conditions. Lag times of 37 days (sulfate amended) to more than 100 days (methanogenic) were observed prior to activity.